scholarly journals Serotype-dependent recombinant adeno-associated vector (AAV) infection of Epstein–Barr virus-positive B-cells, towards recombinant AAV-based therapy of focal EBV + lymphoproliferative disorders

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Elham Ahmadi ◽  
Mehrdad Ravanshad ◽  
Jun Xie ◽  
Rajesh Panigrahi ◽  
Sandeep S. Jubbal ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Lymphoproliferative Disorders ◽  
Cell Tumor ◽  
Barr Virus ◽  
Human B Cells ◽  
Epstein Barr ◽  
Increased Susceptibility

Abstract Background B-cell proliferative disorders, such as post-transplant lymphoproliferative disease (PTLD), are increased among persons afflicted by T-cell compromise. Most are Epstein–Barr virus (EBV) + and can first present with a focal lesion. Direct introduction of oncolytic viruses into localized tumors provides theoretical advantages over chemotherapy, immunotherapy and radiation therapy by reducing systemic toxicity. Despite extensive study as a vehicle for gene therapy, adeno-associated viruses (AAV) have rarely been applied to human cancer research due to technical and theoretical obstacles. Moreover, human B-cells have historically been described as resistant to AAV infection. Nonetheless, advances using different recombinant (r)AAV serotypes with unique tropisms to deliver cytotoxic therapy suggested a localized anti-tumor approach was feasible. Methods As a prelude to the development of a therapeutic vehicle, the ability of fifteen distinct EGFP-bearing rAAV serotypes to transduce human B-cells, including primary, immortalized, and B-cell tumor lines ± EBV was assessed by confocal microscopy, flow cytometry and subsequently cell viability assay. Results Rank order analysis revealed augmented transduction by rAAV6.2 and closely related virions. EBV infection of EBV-negative B-cell tumor lines and EBV immortalization of primary B-cells increased susceptibility to rAAV6.2 transduction. As a proof of concept, transduction by rAAV6.2 encoding herpes simplex virus type 1 (HSV1)-thymidine kinase (TK) eliminated TK-negative rhabdomyosarcoma cells and diminished viability of transduced B-cell lines upon incubation with ganciclovir. Conclusions rAAV serotypes differentially transduce human B-cell lines reversing the dogma that human B-cells are refractory to AAV infection. EBV + B-cells display increased susceptibility to rAAV6.2 infection, uncovering a new method for improved nucleic acid transfer into transfection-resistant B-cell lines. The introduction of a functional suicide gene into the rAAV6.2 genome identifies a candidate vector for the development of rAAV-based oncolytic therapy targeting focal EBV-bearing B-lymphoproliferative disorders.

Quantification of C3dg/Epstein-Barr Virus Receptors on Human B Cells and B Cell Lines

Complement ◽  
1988 ◽  
Vol 5 (2) ◽  
pp. 98-107 ◽  
Author(s):  
J. Møller Rasmussen ◽  
H.V. Marquart ◽  
R. Rask ◽  
H.H. Jepsen ◽  
S.-E. Svehag
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Human B Cells ◽  
Virus Receptors ◽  
Epstein Barr

scholarly journals Use of Epstein-Barr virus-transformed B cell lines for the generation of immunoglobulin-producing human B cell hybridomas.

1982 ◽  
Vol 156 (3) ◽  
pp. 930-935 ◽  
Author(s):  
N Chiorazzi ◽  
R L Wasserman ◽  
H G Kunkel
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Monoclonal Antibodies ◽  
Barr Virus ◽  
Human B Cells ◽  
Series Of Experiments ◽  
Epstein Barr ◽  
Human B Cell

HGPRTase-deficient EBV-transformed B cell lines were shown to be effective fusion partners with mitogen-activated human B cells for the construction of Ig-producing human B cell hybridomas. In a series of experiments using these lines and B cells from several tissue sources, approximatley 20% of the cultures plated were consistently positive for growth after hypoxanthine-aminopterin-thymidine selection and approximatley 30% of these synthesized significant new Ig. A marked increase in Ig secretion was observed after hybridization, which was due to new Ig; Ig from the parental lime was shown to disappear in several instances. Special analyses were carried out on a human hybridoma secreting antibody specific for tetanus toxoid and tetanus toxin and stable subclones were derived. These studies suggest that EBV-transformed lines will prove useful in human hybridization studies, thus making a large library of B cell lines available for the generation of human monoclonal antibodies.

scholarly journals Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Lisa Grossman ◽  
Chris Chang ◽  
Joanne Dai ◽  
Pavel A. Nikitin ◽  
Dereje D. Jima ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Cellular Aggregation ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out. Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

scholarly journals Rewiring of B cell receptor signaling by Epstein–Barr virus LMP2A

2020 ◽  
Vol 117 (42) ◽  
pp. 26318-26327
Author(s):  
Kamonwan Fish ◽  
Federico Comoglio ◽  
Arthur L. Shaffer ◽  
Yanlong Ji ◽  
Kuan-Ting Pan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Barr Virus ◽  
Human B Cells ◽  
Bcr Signaling ◽  
Epstein Barr

Epstein–Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

scholarly journals Compatibility of the gH homologues of Epstein–Barr virus and related lymphocryptoviruses

2007 ◽  
Vol 88 (8) ◽  
pp. 2129-2136 ◽  
Author(s):  
Liguo Wu ◽  
Lindsey M. Hutt-Fletcher
Keyword(s):  
B Cells ◽  
Epithelial Cells ◽  
B Cell ◽  
Cell Fusion ◽  
Epstein Barr Virus ◽  
Common Marmoset ◽  
Barr Virus ◽  
Human B Cells ◽  
The Common ◽  
Epstein Barr

Glycoprotein gH, together with its chaperone gL and a third glycoprotein gB, is essential for cell–cell fusion and virus–cell fusion mediated by herpesviruses. Epstein–Barr virus (EBV), the prototype human lymphocryptovirus, requires a fourth glycoprotein gp42 to support fusion with B cells in addition to epithelial cells. Two other lymphocryptoviruses, the rhesus lymphocryptovirus (Rh-LCV) and the common marmoset lymphocryptovirus (CalHV3), have been sequenced in their entirety and each has a gp42 homologue. Combinations of proteins from EBV, Rh-LCV and CalHV3 were able to mediate fusion of epithelial cells, but, even when complexed with EBV gp42, only Rh-LCV and not CalHV3 proteins were able to mediate fusion with human B cells. CalHV3 gL was also unable to function effectively as a chaperone for EBV or Rh-LCV gH. The Rh-LCV gH homologue supported more fusion than EBV gH with an epithelial cell and supported the highest levels of fusion with a B cell. Chimeric constructs made from Rh-LCV gH and EBV gH that have 85.4 % sequence identity should prove useful for mapping the regions of gH that are of importance to fusion as a whole and to B-cell fusion in particular.

scholarly journals Epstein-Barr virus transformation of human pre-B cells.

1983 ◽  
Vol 158 (2) ◽  
pp. 616-622 ◽  
Author(s):  
M Hansson ◽  
K Falk ◽  
I Ernberg
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Bone Marrow Cells ◽  
Fetal Bone ◽  
Barr Virus ◽  
Epstein Barr

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in establishment of B lymphoblastoid cell lines that reflect normal B cell phenotypes. In this study we have investigated whether immature B cells from fetal bone marrow and liver can serve as targets for EBV. The fetal bone marrow cells were readily transformed by EBV. Among the resulting cell lines, five were surface Ig (sIg)-negative. Three B cell-associated antigens defined by monoclonal antibodies were expressed to the same extent on the fetal cell lines, whether they belonged to the sIg- or sIg+ group. The various differentiation stages that these cell lines may represent are discussed.

scholarly journals Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

2021 ◽  
Vol 17 (4) ◽  
pp. e1009117
Author(s):  
Ezgi Akidil ◽  
Manuel Albanese ◽  
Alexander Buschle ◽  
Adrian Ruhle ◽  
Dagmar Pich ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Gene Editing ◽  
Barr Virus ◽  
Ebv Infection ◽  
Highly Efficient ◽  
Human B Cells ◽  
Epstein Barr

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.

Epstein-Barr Virus (EBV) LMP2A induces alterations in gene transcription similar to those observed in Reed-Sternberg cells of Hodgkin lymphoma

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 4166-4178 ◽  
Author(s):  
Toni Portis ◽  
Patricia Dyck ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Gene Transcription ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Expression Of Genes ◽  
Epstein Barr

AbstractEpstein-Barr virus (EBV) is associated with the development of a variety of malignancies, including Hodgkin lymphoma. One of the few viral transcripts expressed in EBV-positive Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma is latent membrane protein 2A (LMP2A). This viral protein blocks B-cell receptor (BCR)-signaling in vitro. Furthermore, expression of LMP2A in developing B cells in vivo induces a global down-regulation of genes necessary for proper B-cell development. In this study we have analyzed gene transcription in primary B cells from LMP2A transgenic mice, LMP2A-expressing human B-cell lines, and LMP2A-positive and -negative EBV-infected lymphoblastoid cell lines (LCLs). We demonstrate that LMP2A increases the expression of genes associated with cell cycle induction and inhibition of apoptosis, alters the expression of genes involved in DNA and RNA metabolism, and decreases the expression of B-cell-specific factors and genes associated with immunity. Furthermore, many alterations in gene expression induced by LMP2A are similar to those recently described in HRS cells of Hodgkin lymphoma and activated, proliferating germinal center centroblasts/centrocytes. These correlations suggest that LMP2A expression in EBV-infected B cells may lead to the induction and maintenance of an activated, proliferative state that could ultimately result in the development of Hodgkin lymphoma. (Blood. 2003;102: 4166-4178)

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